1. Field of the Invention
This invention relates to conjugates of anti-HIV nucleosides and antiviral ether and thioether lipids working as both prodrug and combination chemotherapy with two or more antiviral moieties with different target sites, exhibiting enhanced antiviral activity.
This invention also relates combination chemotherapy of micellar formulation of the conjugate and other antiviral drugs.
2. Brief Description of the Background
The human immunodeficiency virus (HIV), an enveloped single-stranded RNA virus, is the severe causative agent of acquired immune deficiency syndrome (AIDS). Efforts to develop drugs and vaccines that combat AIDS have resulted in dozens of potential treatments, which are in clinical and preclinical study. An unprecedented triple challenge is 1) stop the HIV, 2) restore the damaged immune system, and 3) treat any opportunistic infections. In view of the etiologic role of HIV, antiviral agents which inhibit HIV replication can be considered as a viable approach towards an effective chemotherapy of AIDS. Major steps in the replicative cycle of HIV are absorption, penetration, uncoating, reverse transcription, circulation, integration, translation, assembly and budding. For inhibitors of HIV replication, absorption, reverse transcription, transactivation, and budding have been identified or postulated as target. The majority of the anti-HIV agents appears to act at the reverse transcriptase level, and most of them belong to the class of 2',3'-dideoxynucleosides. To date, 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and 2',3'-dideoxycytidine (ddC) are the only compounds licensed for clinical use in the treatment of AIDS In addition to these compounds, various other 2',3'-dideoxynucleoside analogs, 3'-substituted 2',3'-dideoxynucleoside, and 2',3'-didehydro-2',3'-dideoxynucleoside have proven to be potent and selective inhibitors of HIV replication with selectivity All 2',3'-dideoxynucleoside analogs are assumed to act in a similar fashion as AZT, which implies that they need first to be converted intracellularly to their 5'-triphosphates. These dideoxynucleoside 5'-triphosphates can compete with normal 2' -deoxynucleotides for binding to reverse transcriptase or they can be incorporated into the growing DNA chain to bring about termination of viral DNA synthesis. And 2',3'-dideoxynucleoside 5'-triphosphates have higher affinities for HIV reverse transcriptase than for cellular DNA polymerase .alpha.. If, in fact, the 2',3'-dideoxynucleosides are degraded by catabolic enzymes, i.e. deaminases or phosphorylases, before being anabolized by the kinases, they may never reach the 5'-triphosphate stage.
Membrane-active ether lipid analogs of platelet-activating factor selectively inhibit HIV-1 plaque formation (IC.sub.50 0.2-1.4 .mu.M) and the inhibition correlates with inhibition of infectious HIV-1 production, which indicate that the ether lipid analogs inhibit both HIV-induced cytopathology and infectious virus multiplication by inducing defective intracytoplasmic vacuolar HIV-1 formation in T-cells. Being membrane interactive, the ether lipid analogs are ideally suited for combination chemotherapy with DNA-interactive anti-HIV nucleoside analogs. The combination of ether lipid analog and AZT produced an apparent synergistic action in suppressing infectious HIV-1 replication (Kucera, et al., AIDS Research and Human Retroviruses 6, 491, 1990).
In order to facilitate the mechanism of action of antitumor nucleoside, 1-.beta.-D-arabinofuranosylcytosine (ara-C), and to overcome its degradation by catabolic enzymes, a series of new lipophilic biotransformed prodrugs of ara-C were synthesized by covalently conjugating ara-C with phosphatidic acids via a naturally occurring phosphate linkage. These liponucleotides include the ara-C conjugates of 1,2-diacyl (Hong, C. I. et al., Cancer Drug Delivery 1, 181, 1984), 1-O-alkyl (ether) (Hong, C. I. et al., J. Med. Chem. 29, 2038, 1986), and 1-S-alkyl (thioether) (Hong, C. I. and West, C. R., U.S. Pat. No. 4,622,392, Issued Nov. 11, 1986) phospholipids. These conjugates have ara-C substituted for cytidine or both ara-C and ether (or thioether) lipid substituted for cytidine and diacylglyceride in cytidine diphosphate diacylglyceride, a precursor for membrane phosphatidylinositol and cardiolipin. The rationale is that the conjugates are not only prodrugs of ara-C but also may generate two cytotoxic principles (ara-CTP and ether or thioether lipid) with different target sites, nucleic acid synthesis and membrane, inside a neoplastic cell. Most of the ara-C conjugates produce significant increases in the life span and long-term survival of mice with L1210 and P388 leukemia, independent of treatment schedule. Among them, ara-CDP-DL-PTBA (hereinafter referred to as Cytoros), a conjugate of thioether phospholipid, is the most effective of those tested (Hong, C. I. Drugs of the Future 15, 245, 1990). Cytoros showed considerably higher efficacy than ara-CDP-L-dipalmitin, conjugate with 1-O-acyl phospholipid, against myelomonocytic WEHI-3B leukemia in mice. Cytoros has a rather broad spectrum of therapeutic activity in mice with various solid tumors such as colon 26 carcinoma, M5076 sarcoma, Lewis lung carcinoma, and C-1300 neuroblastoma. Furthermore, this drug displayed antimetastic activity in mice with M5076 sarcoma and Lewis lung carcinoma. Cytoros showed a strong antitumor activity against human colorectal cancer xenografts and PSN-1 human pancreatic tumor in nude mice. Cytoros alone and in combination with VP-16 markedly retarded both primary and lymph node metastasis of rat mammary carcinoma SMT-2A. Thus, Cytoros seems to be not only highly active by itself, but also serves as a powerful and selective drug delivery system for the eradication of metastatic cancer. The mode of action of Cytoros and other ara-C conjugates of phospholipids remains largely unknown. They may act as lipophilic prodrugs with sustained release of ara-C or facilitate transfer of the active moiety across tumor cell membranes because of their lipophilic nature. Evidence from the studies with L1210 leukemia in mice demonstrated that the administration of Cytoros led to more prolonged plasma ara-C concentration and intracellular retention of ara-CTP than those resulting from ara-C. This may account for its increased therapeutic effectiveness, since it has been shown that increased leukemic cell retention of ara-CTP correlates with therapeutic response. Further, other possible pharmacologically favorable properties of the conjugates as compared to the parent drug include resistance to hydrolysis by cytidine deaminase, rapid interaction with serum lipoproteins, rapid uptake by cells, and effects on lipid biosynthesis. Cytoros showed a selective cytotoxicity to L1210 leukemic cells compared to normal mouse bone marrow cells.
Previously, anti-HIV nucleoside conjugates of phospholipids (diacyl, ether thioether, and amido) have been reported. Dimyristoyl-phosphatidylAZT, dimyristoylphosphatidylddT, dimyristoylphosphatidylddC, and AZT dipalmitoylglycerol were encapsulated in liposomes and these liposomal liponucleotides were shown to have antiretroviral activity in HIV-infected U937 (IC.sub.50 0.2-12 .mu.M) and CEM cells (IC.sub.50 1.7-22 .mu.M) (Hostettler, K. Y. et al., J. Biol. Chem. 265, 6112, 1990). AZT and ddI conjugates of amidoalkyl, oxyalkyl, and thioalkyl ether lipids through phosphate and phosphonate linkages showed promising in vitro activity (IC.sub.50 0.02-1.56 .mu.M) with 5-10 fold reduction in cell cytotoxicity compared to AZT alone (Piantadosi, C. et al., J. Med. Chem. 34, 1408, 1991). Hostettler's conjugates are those with non-antiretroviral diacylglycerides with phosphodiester or pyrophosphate diester bond. Thus, these conjugates are considered to be simple prodrugs of anti-HIV nucleosides. Since the majority are conjugates with a phosphodiester (monophosphate) bond, they cannot form micelles by sonication; therefore, they must be encapsulated in liposomes for testing. Also, Piantadosi's conjugates contain monophosphate or phosphonate bonds which cannot be formulated in micellar solution by sonication.